Although significant progress in structure-based vaccine design has been made, improvement in antigen design would have a significant effect in improving the immunogenicity of this vaccine.
Given that the spike is highly metastable and often requires modifications, the addition of prolines has been demonstrated to be one mechanism that can be employed to achieve stability [269]. The study showed that adding prolines to stabilize the spike protein in its prefusion conformation was largely advantageous for inducing stronger immune responses. This strategy has been employed by several others, and has recently been modified by Schaub and colleagues to include six prolines instead of two [270]. The authors demonstrated that the large and heavily glycosylated spike protein was difficult to stabilize in its prefusion conformation, and this was remedied by the addition of four prolines to form the HexaPro design [270].
Overall improvement of the MVA-SARS2-SΔTM and other vaccines is necessary to accommodate the evolving SARS-CoV-2 virus. It is possible that the inclusion of other important antigens such as the nucleocapsid protein could result in a more immunogenic vaccine. Coexpression with the highly expressed nucleocapsid, or generation of nucleocapsid-only expressing vaccines is worth exploring [271], [247], [272]. Due to the fact that it is relatively more stable as compared to the spikeand plays a crucial role in viral replication, the protein is a target for cytotoxic T-cells [273].
This has been demonstrated by several others and is being shown as a viable target [274], [275],
108
[272]. Studying T-cells and their role in mitigating severe disease has proven challenging [276]
yet it is crucial to understand this for design of next generation vaccines. Given the frequently mutating virus and high levels of breakthrough infections, T-cell immunity has been shown to be important because T-cells have the ability to recognize parts of SARS-CoV-2 that are not frequently mutating such as the nucelocapsid [277]. Additionally, it has been demonstrated that severe and fatal COVID-19 disease is characterized by lymphopenia and high levels of the biomarker CXCL10, which were shown to be strongly positively correlated with T-cell death when compared in ICU and non-ICU patients [278]. These findings together elucidate the role of T-cells which is critical as the virus continues to evade host immunity. Further investigations could be done to determine the effect of including a shorter length of our designed antigen, with either the S1 only or S2 only subunits. Kim et al., 2021 demonstrated that the S1 subunit alone given at high dose produced high titres of neutralizing antibodies. However, when compared to a low-dose of a full-length spike vaccine, the S1 subunit produced lower neutralizing antibodies [279]. It is fundamental for vaccines to be “updated” through employment of strategies in order to continue offering protection to humans. Considering the continuing COVID-19 pandemic, vaccine development is an ongoing process. The mutation of the SARS-CoV-2 virus presents an additional challenge which shifts vaccine development to focusing on the effect of already approved and administered vaccines on emerging variants. Booster shots are in discussion with some countries already administering these for several vaccines [280], [281], [282]. The timing of the booster shots and questions of whether to get the same shot or a different vaccine as a booster are all under consideration with the aim of eliciting as safe, strong, broad and long-lasting immune responses as possible.
Combining vaccines expressing different immunogenic antigens has been shown to induce both T-cells and antibodies [235]. A possible application of this would be the combination of the MVA- SARS2-SΔTM vaccine with another vaccine, such as priming with the S protein and boosting with another antigen e.g. the RBD. This approach has been explored whereby it was shown that priming with the S protein and boosting with either the S or the RBD was highly immunogenic. On the other hand, this study observed that priming with the RBD and boosting with the S or the RBD was not immunogenic [283].
109
Once further work has been done to improve immunogenicity of the MVA-SARS2-SΔTM vaccine, evaluations on dose safety would be necessary to determine the toxicity of the vaccine.
This would involve varying doses to conclude on adverse effects and what the “right” dose is.
Hamster challenges will be set up where hamsters will be vaccinated and subsequently challenged with variants of the SARS-CoV-2 virus. Following this, hamsters will be assessed for histopathological changes, viral load and weight changes to evaluate the vaccine’s ability to provide protection against severe infection.
APPENDICES
APPENDIX A: REAGENT FORMULATIONS
110 Luria Bertani agar (100ml)
Tryptone 1 g
Yeast extract 0.5 g
NaCl 0.5 g
Agar 1.5 g
Water Adjust to 100 ml
10% (v/v) Complete Dulbecco’s Modified Eagle Medium (cDMEM) Dulbecco’s Modified Eagle Medium 450 ml
10% Heat inactivated Fetal Calf Serum 50 ml 1% Penicillin-Streptomycin antibiotic 5 ml
Freezing medium Concentration (v/v)
Heat inactivated Fetal Calf Serum 80%
Dimethyl Sulfoxide (DMSO) 10%
DMEM 10%
4X protein loading buffer
100% Glycerol 4 ml
1.5M Tris/HCl (p.H 6.8) 1.6 ml
SDS 0.8 g
Bromophenol blue 4 mg
β-mercaptoethanol 0.5 ml
ddH2O 3.9 ml
10 X SDS page running buffer
SDS 10 g
Tris 30.3 g
Glycine 29 g
ddH2O adjust to 1000 ml
10 X transfer buffer
SDS 3.7 g
Tris 58 g
Glycine 144.1 g
ddH2O adjust to 1000 ml
PBST
10 X PBS 100 ml
10% Tween 20 10 ml
ddH2O Adjust to 1000 ml
Block buffer
1 X PBS 100 ml
Instant skim milk power 5 g
10% Tween 20 1 ml
111
4% (v/v) stacking gel (5 ml)
40% Acryl-bisacrylaminde mix 0.5 ml
1.5 M Tri (pH 6.8) 0.63 ml
10% SDS 50 µl
10% APS 50 µl
TEMED 5 µl
ddH2O 3.8 ml
8% (v/v) resolving gel (10 ml)
40% Acryl-bisacrylaminde mix 2 ml
1.5 M Tri (pH 8.8) 2.5 ml
10% SDS 0.1 ml
10% APS 0.1 ml
TEMED 6 µl
ddH2O 5.3 ml
2% Bovine Serum Albumin (BSA)
BSA 2 g
1 X PBS 100 ml
4 % Paraformaldehyde
Paraformaldehyde powder 40 g
1 X PBS 800 ml
Red Blood Cell Lysis buffer
NH4CL 8.34 g
EDTA 0.037 g
NHCO3 1 g
ddH2O 1000 ml
Resuspension Solution P1
Tris-HCL (1 M, pH 8.0) 2.5 ml
20% (w/v) glucose 0.91 g of glucose in 4.55 ml of ddH2O
EDTA (0.5 M, pH 8.0) 2 ml
ddH2O 0.95 ml
RNase (200 µg/ml) 400 µl
Lysis buffer P2
Sodium hydroxide (200mM) 8 g in 900 ml of ddH2O
1% (v/v) SDS 100 ml of 10% SDS in 1 L of ddH2O
112 Neutralization buffer P3 (100 ml)
Potassium acetate (3.0 M, pH 4.8) 4.41 g
Glacial acetate 7.5 ml
ddH2O adjust to 15 ml
113 APPENDIX B: pMVA-FNK2 Sequence
G1L-SARS-CoV-2 SΔTM-GGTGGACCGGTAAGCTTCT-mH5- CCGGGTTATAG -K1L- CGTGGCGGTCGACTCTAA-pSS- GAGCTCAGAAAAA-eGFP- GGT- p7.5- ATCGA-I8R
CTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGC TCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAG ACCGAGATAGGGTTGAGTGGCCGCTACAGGGCGCTCCCATTCGCCATTCAGGCTGC GCAACTGTTGGGAAGGGCGTTTCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCG AAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTC ACGACGTTGTAAAACGACGGCCAGTGAGCGCGACGTAATACGACTCACTATAGGGC GAATTGTGCGAAGGCCGTCAAGGCCTAGGCGCGCCTGTCGACGCCGGCGCATGCAC TAGTGTTCTTGACGCAACCAATGATGGACTGATTAAGAAACCTTATAGAAGTATACC CCTAATGAAGCGTCTAACATCTAATGAAATATTTATACGATACGGAGACGCGTCTCT CATGGACATGATAACTTTATCATTGTCTAAACAAGATATATCATTAAAAAGAAATGC CGAAGGAATACGTGTAAAACATAGTTTTTCAGCTGATGATATACAGGCAATTATGGA ATCTGATTCGTTTTTAAAGTATAGTAGATCAAAACCAGCTGCGATGTATCAATATAT ATTTCTATCATTTTTTGCTAGTGGTAATTCCATAGATGACATATTGGCAAATAGAGAT TCTACCTTAGAATTTTCTAAAAGAACTAAAAGTAAAATTTTGTTTGGTAGGAATACC AGATACGACGTCACTGCAAAATCTAGTTTTGTATGTGGTATAGTACGAGGTAAATCA TTGGATAAAACGTCTCTGGTTGAAATGATGTGGGATCTCAAGAAGAAAGGATTAAT ATATTCTATGGAATTTACCAATCTATTGAGCAAGAATACCTTTTATCTGTTCACATTT ACTATCTACACTGATGAAGTATACGATTATCTAAACACTAATAAACTTTTTTCTGCA AAATGTTTAGTCGTGTCTACAAAAGGAGATGTGGAAAATTTTTCATCTCTAAAAAAA GATGTGGTCATTAGAGTTTGAGAATTCTCAGTGGTGGTGGTGATGGTGCCCGCTGCC CCCGCTGCCGCCGCTCCCTCCCAGGAAGGTGCTCAGCAGCACCCACTCGCCATCCTT TCTCACGTAGGCCTGTCCGTCCCGAGGTGCCTCTGGGATATATCCGGAGCCTGGAGA GCCGGAGCCggatccAGGCCACTTGATGTACTGCTCATACTTGCCCAGCTCCTGCAGAT CGATCAGGCTCTCGTTCAGATTCTTGGCCACCTCGTTCAGTCTGTCGATCTCCTTCTG GATGTTCACCACGCTGGCATTGATGCCAGAGATGTCGCCCAGATCCACGTCGGGGG AGGTGTGATTCTTGAAGTACTTATCCAGCTCCTCCTTAAAGCTGTCCAGCTCTGGCTG CAGAGGATCATACACGGTATTGTTCACGATGCCGATGACCACGTCACAGTTGCCGCT CACGAAGGTATTGTCTGTGGTGATGATCTGGGGCTCGTAGAAATTTCTCTGTGTCAC AAACCAGTGGGTGCCGTTGGACACGAACACGCCCTCCCGTGGAAAGTGGGCCTTGC CATCGTGGCAGATGGCAGGGGCTGTGGTGAAGTTCTTCTCCTGGGCGGGCACGTAG GTCACGTGCAGAAACACCACTCCGTGTGGGGCGGACTGTGGGAAGCTCATCAGGTG ATAGCCCTTGCCACAAAAGTCCACCCTCTTGGACTGTCCCAGCACGCACTCGCTCAT CTTGGTTGCTGCCAGATTGGCGCTTGCCCTGATCTCTGCTGCCCTGATCAGCTGCTGT GTCACGTAGGTCTGCAGAGACTGCAGTCTGCCTGTGATCAGCCGGTCGATCTGCACC TCGGCCTCCACCTTGTCCAGCCTGGACAGGATATCATTCAGCACGCTAGAGATGGCG CCGAAGTTGGAGCTCAGCTGCTTCACCAGGGTATTCAGGGCCTGGGCGTTCTGATTC ACCACATCCTGCAGCTTGCCCAGGGCGGAGGCTGTAGAGGACAGGCTGTCCTGGAT CTTGCCGATGGCGCTGTTAAACTGATTGGCGATCAGCTTCTGGTTCTCGTACAGCAC ATTCTGGGTCACGCCGATGCCGTTGAAGCGATAGGCCATCTGCATGGCAAAGGGGA
114
TCTGCAGGGCGGCTCCTGCGCCGAAGGTCCATCCGCTTGTGATGGTTCCTGCCAGCA GGGCAGATGTGTACTGGGCGATCATCTCATCTGTCAGCAGTGGAGGCAGCACGGTC AGGCCATTAAACTTCTGGGCACAGATCAGGTCGCGGGCTGCGATGTCGCCCAGGCA ATCGCCATACTGCTTGATGAAGCCGGCATCGGCCAGGGTCACCTTGTTGAACAGCAG GTCCTCGATAAAGCTCCTCTTAGAAGGCTTGGAAGGATCGGGCAGGATCTGGCTGA AGTTGAAGCCGCCAAAGTCCTTGATGGGTGGGGTCTTGTAGATCTGCTTCACCTGGG CGAACACCTCCTGTGTGTTCTTATCCTGCTCCACTGCGATGCCTGTCAGGGCCCTATT CAGCTGGGTACAAAAGGAGCCGTACTGCAGCAGCAGGTTAGAGCACTCGGTGGAAT CGCCACAGATATACATTGTGCAGTCCACGGATGTCTTGGTCATGCTCACGGGCAGGA TCTCTGTGGTCACGCTGATTGTGAAGTTGGTTGGGATGGCGATGCTATTGTTAGAGT AGGCCACGCTGTTCTCGGCGCCCAGGGACATGGTATAGGCGATGATAGACTGGGAT GCCACGCTTCTCCGGCGCCTTCTCCGTGGAGAGTTTGTCTGGGTCTGGTAGGAGGCA CAGATGCCGGCGCCGATTGGGATGTCGCACTCATAAGAATTGTTCACGTGCTCTGCT CCGATCAGGCATCCGGCGCGTGTCTGGAACACGTTGGAGCCGGTGCTGTACACCCTC CATGTTGGGGTCAGCTGATCGGCGTGGATGGCCACAGGCACCTCGGTACAATTCACG TCCTGATACAGCACGGCCACCTGGTTGGATGTATTGGTGCCGGGTGTGATCACAGAC ACGCCGCCGAAGGAGCAGGGTGTGATATCCAGGATCTCCAGGGTCTGGGGGTCCCG CACGGCGTCTGTGGTATCTGCGATGTCCCGGCCGAACTGCTGAAATGGCAGGAACTT CTTGTTGCTCTCGGTCAGCACGCCTGTGCCGGTCAGGCCGTTGAAGTTGAAGTTCAC GCACTTGTTCTTCACCAGATTGGTGGACTTCTTAGGTCCGCACACTGTTGCTGGTGCG TGCAGCAGCTCAAAGCTCAGCACCACCACTCTGTAAGGCTGATAGCCCACGCCGTTG GTGGGCTGGAAGCCGTAAGACTGCAGTGGGAAATAACAGTTAAAGCCCTCCACGCC ATTGCAAGGGGTAGAGCCGGCCTGGTAGATCTCTGTGGAGATGTCCCGCTCGAAGG GCTTCAGATTGGACTTTCTAAACAGCCGGTACAGATAATTGTAGTTGCCGCCCACTT TGCTATCCAGATTGTTAGAGTTCCAGGCGATCACGCAGCCGGTGAAATCGTCTGGCA GCTTATAATTGTAGTCGGCGATCTTGCCTGTCTGTCCAGGTGCGATCTGGCGCACCTC GTCGCCCCTGATCACGAAAGAATCGGCGTACACGTTGGTAAAGCACAGGTCATTCA GCTTTGTGGGGGACACGCCATAGCACTTAAAGGTGCTGAAAGAGGCGGAGTTGTAC AGCACGCTATAGTCGGCCACGCAGTTAGAGATGCGCTTCCTATTCCAGGCGTACACG CTGGCGAATCTGGTGGCGTTGAACACCTCGCCAAAGGGGCACAGGTTTGTGATATTT GGAAATCTCACGATGGACTCGGTTGGCTGCACCCGGAAATTGCTTGTCTGATAGATG CCCTTCTCCACGGTAAAGCTCTTCAGTGTACACTTTGTCTCAGACAGGGGGTCCAGT GCGCAATCCACTGCGTCTGTGATGGTGCCATTCTCGTTGTACTTCAGCAGGAAGGTC CGAGGCTGCAGATAGCCCACATAGTAGGCTGCTGCTCCTGCGGTCCATCCGCTAGAG GAGTCGCCTGGTGTCAGGTAGGAGCGGTGCAGGGCCAGCAGTGTCTGAAACCTGGT GATGTTGATGCCGATTGGCAGATCCACCAGAGGCTCCAGGGCGCTGAAGCCCTGAG GCAGGTCCCGCACCAGGTTGATGGGGGTGTGCTTAGAGTAGATCTTGAAGTAGCCAT CGATATTCTTAAACACGAACTCCCGCAGGTTCTTGAAATTGCCCTGCTTGCCCTCCA GGTCCATCAGGAAAGGCTGGCTCACGTACTCAAATGTGCAATTGTTGGCGCTAGAAT ACACCCTAAACTCGCTCTCCATCCAAGACTTATTGTTCTTGTGATAGTACACGCCCA GGAAGGGATCATTACAAAACTGGAACTCGCACACCTTGATGACCACGTTGGTGGCA TTGTTCACGATCAGCAGGGACTGTGTCTTGCTGTCCAGTGTGGTGCCAAAGATCCAG CCGCGGATGATGTTAGACTTCTCGGTGGAGGCGAAGTACACGCCATCGTTAAAGGG CAGCACTGGATTGTCGAACCTCTTTGTGCCATTGGTGCCGGACACGTGGATGGCGTG GAACCAGGTCACGTTAGAAAAGAATGGCAGAAACAGATCCTGTGTGGAGTGCAGCA CGGAGCTGCGAAACACCTTGTCAGGATAGTACACGCCCCTTGTGAAGCTATTGGTAT
115
AGGCAGGGGGCAGCTGAGTTCTGGTAGTCAGGTTCACGGGGCTCACAAACACGGCT CCGCACAGCAGCAGCACGCAGCACAGTCCTCTTTTCATAGCATCCATGGTGGACCGG TAAGCTTCTAGATATTTATGATTATTTCTCGCTTTCAATTTAACACAACCCTCAAGAA CCTTTGTATTTATTTTCAATTTTTCCCGGGTTATAGGATATTGATTCTTCTAATGAAA ATCTTTAGAATGAATAGAGATATGTTAATAACAGGGTTGTTAGCTACACCCATTTTT GTAGCTAGTTGTTCACAATCGGGCCCGTCGACAGAAAAATTAGTTCTTCTTTACACA GTTTACGTACATAAGTCTTAGTTCTTTGTTCTTAGAGATGATCTCATCCAACTTGTTG TTCTTTACGATATCCAAATCCTTTGTGTAAGAATCAGACTTGTATTCTACATGTTTTT CGATGATCATCTTCGCGATTTCCGCATCATCCAATAGTACGTTTTCTAGGTTCGCAGA GTAGATGTTGATATCGTACTTAAACAAGGCTTGTAGCATTTCGATATCCTTGTTATCA ATCGCTAGCTTGATATCCGGGATAAACAACAAACTGTTGTTTGTGTTTGTAGATGTC ATGTAATCTAGTAGTAGGATCATCATATCTACATGTCCGTTCTTGATTGTGATATGGA TACAAGATAGTAGGATCGCTAGATCAAATGTAGACGGGATTTCAGATAGAAAGTAA GATACGATAGATACATCGTTTAGCATTACGGCATGGTAAAAAGATGTTTTCCATCCT GTCTTTCCGTAAAACATTAGTCTCCAATTTTTCTTAACAAATAGCTTTACTGTTTGCA TGTTTCCAGAATCTACCGCGTAGTACAACGCTGTGTTTCCCTTATCATCAAATTGACT GTCGTCTAAACCAGAAAACAACAGGATTTTCACAATCTTAGTGTCCTCTAGTGTAGC AGCTTGGTGCAGAGGGAACTCGTTTTCCAATAAGTTCTTTAGAGCACCTGCATTCAG TAGAGTGCAGACCAATCGTACATTGTTGTCTGCAATAGCGTAGTAACTAGCTGAGTG ACCGTGTACGTCAGCTTTGAAAGTGTCCTTAGAACTTAAGAAAGACTTCAATTGTTT ACTTTTCCATGTGTTGATTCTACTCAAATCCATCGTGGCGGTCGACTCTAATATTTAT ATTCCAAAAAAAAAAAATAAAATTTCAATTTTGAGCTCAGAAAAATTACTTGTACAG CTCGTCCATGCCGAGAGTGATCCCGGCGGCGGTCACGAACTCCAGCAGGACCATGT GATCGCGCTTCTCGTTGGGGTCTTTGCTCAGGGCGGACTGGGTGCTCAGGTAGTGGT TGTCGGGCAGCAGCACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGGTAGTGGTCG GCGAGCTGCACGCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTCACCTTGATG CCGTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGTAGTTGTACTCCA GCTTGTGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAGCTCGATGC GGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGCGCGGGTCTTGTAGTTGCCGT CGTCCTTGAAGAAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGA AGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAG GTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGAT GAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCCTCGCCGGACACGCT GAACTTGTGGCCGTTTACGTCGCCGTCCAGCTCGACCAGGATGGGCACCACCCCGGT GAACAGCTCCTCGCCCTTGCTCACCATGGTCATTCGACGGGATCCGTCACTGTTCTTT ATGATTCTACTTCCTTACCGTGCAATAAATTAGAATATATTTTCTACTTTTACGAGAA ATTAATTATTGTATTTATTATTTATGGGTGAAAAACTTACTATAAAAAGCGGGTGGG TTTGGAATTAGATCGATTTAGTTATTATCTACAGGAACAAATATAGTATCTGAAATC ATATTCATATATCCCGTTAGAGGTCTATGATAATATATAGTAGCGTTTGTTCCGTTAT AGACACCGAATAATATTTTACAAAAGTGTATATACGTATCATCATCTTTATGTTTAA AATTTAAAATCTTAATTCGTAAATTTAGAGATAAAATGGCTTCTTGTACAATACTTGT TAATTCTCCCGTCCTCTCAAAATTATCCAACTCCTCAGCGAGAATAGGACTTAGTAC ATAAAGTTTAGCATACTCTATCATCTTCATATAATAATTGGATAATAATTTATTCCAT GTTTCTGTACTAATATCGAACGAGTCTATATATTCCTTTGTACGCCATAGAATATCCA AATTTGTAGGGATTATAAACAAATCTTCGGGGAGTGTTAGATTAAACTTATTAGCGT ACAATATATAATTATGTAGAAATTCTGAATCTATTCGCTGTATAGACTTCATATAAG
116
ACAGATCATAGAAATATACGTATGTCCCAGGGGTACCGTTAATTAACTGGCCTCATG GGCCTTCGCATCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTA ACATGGTCATAGCTGTTTCCTTGCGTATTGGGCGCTCTCCGCTTCCTCGCTCACTGAC TCGCTGCGCTCGGTCGTTCGGGTAAAGCCTGGGGTGCCTAATGAGCAAAAGGCCAG CAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCG CCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA CAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTG TTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGC GCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAG CTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAAC TATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACT GGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTG GTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAA GCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCG CTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGAT CTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACT CACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTT TAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTG ACAGTTATTACGCACCGCCCTGCCATTCATCACAATACTGCTGCAGTTCGTTCAGCA TACGGCCCACATGAAAGCCATCACACACCGCATGATGCACCTGAATGGCCAGCGGC ATCAGCACTTTATCGCCCTGGGTATAATATTTGCCCATGGTAAACACCGGCGCAAAA AAGTTATCCATGTTCGCCACGTTCAGATCGAAGCTGGTAAAGCTCACCCACGGATTC GCGCTCACAAAGAACATGTTTTCGATGAAGCCTTTCGGAAAATAGGCCAGGTTTTCG CCATAGCACGCCACATCCTGGCTATAAATATGCAGGAACTGGCGAAAATCATCATG ATATTCGCTCCACAGGCTGCTAAAGGTTTCGGTCTGTTCATGAAACACGGTATAGCA CGGATGCACGCTATCCCAAATCACCAGTTCGCCATCTTTCATGGCCATACGAAATTC CGGATGCGCGTTCATCAGACGGGCCAGAATATGAATAAACGCCGGGTAGAATTTGT GTTTGTTTTTTTTCACGGTTTTCAGAAACGCGGTGATATCCAGCTGCACGGTCTGGTT ATAGGTGCACTGCGCCACGCTCTGAAACGCTTCAAAATGTTCTTTACGATGCCACTG GCTAATATCCACGGTGGTATAGCCGGTGATTTTTTTTTCCATACTCTTCCTTTTTCAAT ATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTAT TTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCAC
117 APPENDIX C:
Table 2.5 GenScript SARS-CoV-2-r spike peptide sequence
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLE GKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPC NGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN FNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSY ECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTI SVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQE VFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDC LGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALN TLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPA ICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDP LQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDD SEPVLKGVKLHYT
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